Dependency of cyclic AMP-induced insulin release on intra- and extracellular calcium in rat islets of Langerhans.

Calcium and cyclic AMP are important in the stimulation of insulin release. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) raises islet cAMP levels and causes insulin release at nonstimulatory glucose concentrations. In isolated rat pancreatic islets maintained for 2 d in tissue culture, the effects of IBMX on insulin release and 45Ca++ fluxes were compared with those of glucose. During perifusion at 1 mM Ca++, 16.7 mM glucose elicited a biphasic insulin release, whereas 1 mM IBMX in the presence of 2.8 mM glucose caused a monophasic release. Decreasing extracellular Ca++ a monophasic release. Decreasing extracellular Ca++ to 0.1 mM during stimulation reduced the glucose effect by 80% but did not alter IBMX-induced release. Both glucose and IBMX stimulated 45Ca++ uptake (5 min). 45Ca++ efflux from islets loaded to isotopic equilibrium (46 h) was increased by both substances. IBMX stimulation of insulin release, of 45Ca++ uptake, and of efflux were not inhibited by blockade of Ca++ uptake with verapamil, whereas glucose-induced changes are known to be inhibited. Because IBMX-induced insulin release remained unaltered at 0.1 mM calcium, it appears that cAMP-stimulated insulin release is controlled by intracellular calcium. This is supported by perifusion experiments at 0 Ca++ when IBMX stimulated net Ca++ efflux. In addition, glucose-stimulated insulin release was potentiated by IBMX. These results suggest that cAMP induced insulin release is mediated by increases in cytosolic Ca++ and that cAMP causes dislocation of Ca++ from intracellular stores.     

Evidence for the Involvement of Na/Ca Exchange in Glucose-induced Insulin Release from Rat Pancreatic Islets

Glucose-induced inhibition of Ca⁺⁺ extrusion from the β-cell may contribute to the rise in cytosol Ca⁺⁺ that leads to insulin release. To study whether interference with Na/Ca exchange is involved in this inhibition the effects of glucose were compared to those of ouabain. This substance inhibits Na/K ATPase, decreases the transmembrane Na⁺ gradient in islets, and thus interferes with Na/Ca exchange. Collagenase isolated rat islets were maintained for 2 d in tissue culture with a trace amount of ⁴⁵Ca⁺⁺. Insulin release and ⁴⁵Ca⁺⁺ efflux were then measured during perifusion. In Ca⁺⁺-deprived medium (to avoid changes in tissue specific radioactivity) 16.7 mM glucose inhibited ⁴⁵Ca⁺⁺ efflux. Initially 1 mM ouabain inhibited ⁴⁵Ca⁺⁺ efflux in a similar fashion, the onset being even faster than that of glucose. The effects of 16.7 mM glucose and ouabain were not additive, indicating that both substances may interfere with Na/Ca exchange. In the presence of Ca⁺⁺, 16.7 mM glucose induced biphasic insulin release. Ouabain alone caused a gradual increase of insulin release. Again, the effects of ouabain and 16.7 mM glucose were not additive. In contrast, at a submaximal glucose concentration (7 mM) ouabain enhanced both phases of release. An important role for Na/Ca exchange is suggested from experiments in which Ca⁺⁺ was removed at the time of glucose-stimulation (16.7 mM). The resulting marked inhibition of insulin release was completely overcome during first phase by ouabain added at the time of Ca⁺⁺ removal; second phase was restored to 60%. This could be due to the rapid inhibitory action of ouabain on Ca⁺⁺ efflux thereby preventing loss of cellular calcium critical for glucose to induce insulin release. It appears, therefore, that interference with Na/Ca exchange is an important event in the stimulation of insulin release by glucose.     

The Roles of Intracellular and Extracellular Ca++ in Glucose-Stimulated Biphasic Insulin Release by Rat Islets

Verapamil, an agent known rapidly to block calcium uptake into islets of Langerhans, has been used to study the roles of intra- and extracellular calcium in the two phases of glucose-induced insulin release. Rates of calcium uptake and insulin release during the first phase were measured simultaneously over 5 min in rat islets after maintenance in tissue culture for 2 days. Rates of (45)Ca(++) efflux and insulin release during the first and second phases were also measured simultaneously under perifusion conditions. For this, islets were loaded with (45)Ca(++) during the entire maintenance period to complete isotopic equilibrium. Under static incubation conditions 5 muM Verapamil had no effect upon Ca(++) uptake or insulin release in the presence of 2.8 mM glucose. By contrast, glucose-stimulated calcium influx was totally abolished without there being any significant effect upon first phase insulin release. Thus first phase insulin release is independent of increased uptake of extracellular calcium. The lack of effect of 5 muM Verapamil blockade on first phase insulin release was confirmed, under perifusion conditions, and was in marked contrast to the observed 55% inhibition of second phase release. (45)Ca(++) efflux was inhibited during both phases of the insulin release response.The results show that increased calcium uptake in response to glucose is not involved in the mechanism of first phase insulin release but is required for the full development and maintenance of the second phase release. It seems possible that intracellular calcium is the major regulatory control for first phase insulin release and that intracellular calcium and increased uptake of extracellular calcium contribute almost equally to the second phase of glucose-induced release.     

Somatostatin- and Epinephrine-Induced Modifications of 45Ca++ Fluxes and Insulin Release in Rat Pancreatic Islets Maintained in Tissue Culture

The effects of somatostatin and epinephrine have been studied with regard to glucose-induced insulin release and (45)Ca(++) uptake by rat pancreatic islets after 2 days in tissue culture and with regard to (45)Ca(++) efflux from islets loaded with the radio-isotope during the 2 days of culture. (45)Ca(++) uptake, measured simultaneously with insulin release, was linear with time for 5 min. (45)Ca(++) efflux and insulin release were also measured simultaneously from perifused islets.Glucose (16.7 mM) markedly stimulated insulin release and (45)Ca(++) uptake. Somatostatin inhibited the stimulation of insulin release by glucose in a concentration-related manner (1-1,000 ng/ml) but was without effect on the glucose-induced stimulation of (45)Ca(++) uptake. Similarly, under perifusion conditions, both phases of insulin release were inhibited by somatostatin while no effect was observed on the pattern of (45)Ca(++) efflux after glucose.Epinephrine, in contrast to somatostatin, caused a concentration-dependent inhibition of the stimulation of both insulin release and (45)Ca(++) uptake by glucose. Both phases of insulin release were inhibited by epinephrine and marked inhibition could be observed with no change in the characteristic glucose-evoked pattern of (45)Ca(++) efflux (e.g., with 10 nM epinephrine). The inhibitory effect of epinephrine on (45)Ca(++) uptake and insulin release appeared to be mediated via an alpha-adrenergic mechanism, since is was abolished in the presence of phentolamine.Somatostatin inhibits insulin release without any detectable effect upon the handling of calcium by the islets. In contrast, inhibition of insulin release by epinephrine is accompanied by a partial inhibition of glucose-induced Ca(++) uptake.     

Virus-induced alterations in insulin release in hamster islets of Langerhans.

After the inoculation of Golden Syrian hamsters with the TC-83 vaccine strain of Venezuelan encephalitis (VE) virus, a sustained diminution in glucose-stimulated insulin release and glucose intolerance of shorter duration develops. To understand better the mechanism of this defect in insulin release, we examined insulin secretion in response to several test agents in isolated perifused islets from control and 24-d post-VE virus-infected hamsters. 50 islets were used in all perifusion experiments, and data were expressed as total insulin released as well as peak response for each test agent during a 30-min perifusion period from control and VE-infected islets. After perifusion with 20 mM glucose, a 45% diminution of insulin release was noted in VE-infected islets in comparison with control islets, which in turn was similar to in vivo findings. However, following 1-mM tolbutamide stimulation, insulin release was similar in control and VE-infected islets. In separate studies, 1 mM tolbutamide, 10 mM theophilline, 1 mM dibutyryl cyclic (c)AMP, and 1 mM 8-bromo-cAMP resulted in statistically similar insulin-release curves in control and VE-infected islets. Additional experiments assessing [5-3H]glucose use in control and infected islets after 20 min of perifusion with 20 mM glucose revealed virtually identical values (239 +/- 30-control; and 222 +/- 27-VE-infected islets). Morphological and morphometric evaluation of VE-infected islets (21 d following virus inoculation) showed no changes in islet volume density, beta cell density, and beta cell granulation. Thus, VE virus induces a defect in glucose-stimulated insulin release from hamster beta cells that can be corrected by cAMP analogues and does not alter islet glucose use.     

Role of Ca2+ in secretagogue-stimulated breakdown of phosphatidylinositol in rat pancreatic islets.

Breakdown of phosphatidylinositol (PI) has been shown to be increased during Ca2+-mediated stimulation of cellular responses in many systems and has been proposed to be involved in stimulus-secretion coupling. The effects on PI breakdown of insulin secretagogues that alter cellular Ca2+ or cyclic (c)AMP levels were investigated in perifused rat islets of Langerhans. Isolated islets were labeled with myo-[2-3H(N)]inositol and the efflux of 3H-labeled metabolites was monitored. Glucose (16.7 mM) greatly increased 3H release in a manner that paralleled the second phase of the insulin secretory response; by 60 min, the amount of [3H]PI in the islet decreased by 50%. Removal of Ca2+ from the perifusate or blockade of Ca2+ entry through the voltage-dependent channels by D600 (20 microM) abolished the glucose-induced increase in 3H efflux. Depolarization with 47 mM K+, which increases Ca2+ entry, stimulated protracted 3H and insulin release. Glucose-stimulated output of 3H was not prevented by epinephrine (1 microM) even though the insulin response was abolished. In contrast, 3H output was not affected by isobutylmethylxanthine (1 mM), known to raise cellular levels of cAMP, although insulin release was stimulated. These findings indicate that PI breakdown is not related to the exocytotic process since stimulation of insulin release and PI breakdown could be uncoupled, and that it is not associated with cAMP-mediated regulation of insulin release. PI breakdown in islets differs from the immediate, transient phenomenon reported in other systems in both its timing and requirement for Ca2+. It appears to result from the entry of Ca2+ and not to be the mechanism by which glucose initiates Ca2+ influx.     

Prolonged exposure of human pancreatic islets to high glucose concentrations in vitro impairs the beta-cell function.

The aim of the present study was to clarify whether prolonged in vitro exposure of human pancreatic islets to high glucose concentrations impairs the function of these cells. For this purpose, islets isolated from adult cadaveric organ donors were cultured for seven days in RPMI 1640 medium supplemented with 10% fetal calf serum and containing either 5.6, 11, or 28 mM glucose. There was no glucose-induced decrease in islet DNA content or signs of morphological damage. However, islets cultured at 11 or 28 mM glucose showed a 45 or 60% decrease in insulin content, as compared to islets cultured at 5.6 mM glucose. Moreover, when such islets were submitted to a 60-min stimulation with a low (1.7 mM) followed by a high (16.7 mM) concentration of glucose, the islets cultured at 5.6 mM glucose showed a higher insulin response to glucose than those of the two other groups. Islets cultured at the two higher glucose concentrations showed increased rates of insulin release in the presence of low glucose, and a failure to enhance further the release in response to an elevated glucose level. Islets cultured at 28 mM glucose showed an absolute decrease in insulin release after stimulation with 16.7 mM glucose, as compared to islets cultured at 5.6 mM glucose. The rates of glucose oxidation, proinsulin biosynthesis, and total protein biosynthesis were similar in islets cultured at 5.6 or 11 mM glucose, but they were decreased in islets cultured at 28 mM glucose. These combined results suggest that lasting exposure to high glucose concentrations impairs the function of human pancreatic islets.     

Effects of tacrine on insulin secretion and 86Rb+ and 45Ca++ efflux from rat pancreatic islets.

Tacrine (1,2,3,4-tetrahydro-9-aminoacridine), a drug that has attained interest because of its ability to alleviate symptoms in Alzheimer's type of dementia, was found to stimulate insulin secretion from isolated rat pancreatic islets. The insulinotropic effect of the drug was observed at 8.3 mM but not at 3.3 mM glucose and was dependent on extracellular Ca++. From perifused 86Rb(+)-prelabeled islets, tacrine inhibited the fractional efflux of 86Rb+ at 3.3 mM glucose, but stimulated 86Rb+ efflux at 8.3 mM glucose. These effects persisted in the absence of extracellular Ca++. Tacrine also stimulated 45Ca++ efflux from perifused 45Ca(++)-prelabeled islets at 8.3 mM but had no effect on 45Ca++ efflux at 3.3 mM glucose or in the absence of extracellular Ca++. It is concluded that tacrine potentiates glucose-stimulated insulin secretion by a mechanism that is dependent on extracellular Ca++ and involves an increased Ca++ influx. The increased Ca++ influx is either secondary to a decreased K+ permeability induced by an inhibition of ATP-dependent K+ channels and/or due to a direct effect of tacrine on glucose-activated Ca++ channels.     

Cholinergic Modification of Glucose-Induced Biphasic Insulin Release in Vitro

An in vitro system for perifusion of rat pancreatic islets has been utilized to define the effects of cholinergic agents on the dynamics of insulin release. In the absence of glucose the effects of either acetylcholine or acetyl-beta-methylcholine were minimal at concentrations up to 10(-5) mM. In the presence of low glucose concentration (2.4 mM), both of the muscarinic agents produced dose-dependent biphasic insulin release. Under these conditions significant insulin release was observed over both phases at concentrations of the muscarinic agents as low as 10(-8) mM. Further, the dose response curves relating muscarinic concentration to the total amount of insulin released in each of the two phases showed lack of parallelism between the curves. Nicotinic acid in concentrations up to 10(-5) mM had no effect on insulin release in the presence of 2.4 mM glucose. When the glucose concentration was increased to 16.4 mM, the effects of the muscarinic agents were significantly less than those observed in the presence of 2.4 mM glucose. This held true whether the effect was defined as absolute increment due to the muscarinic agent or as percentage of enhancement. Atropine inhibited insulin release induced by both acetylcholine and by 16.4 mM glucose. These data indicate that cholinergic stimulation can play a significant role in modifying insulin release patterns.     

Fluctuations of calcium, phosphorus, sodium, potassium, and chlorine in single alpha and beta cells during glucose perifusion of rat islets.

To study the relationship between islet hormonal secretion and intracellular content of five elements, a rat islet perifusion technique was used in 24 paired experiments. Control and experimental chambers each containing 100 islets, received 2.8 and 16.7 mM D-glucose, respectively. Effluent was collected frequently for hormone measurements. At eight different time intervals form 0--30 min islets were fixed and prepared for scanning electron microscopy. Over 900 unobscured alpha and beta cells were selected by size and shape criteria. Energy dispersive x-ray analysis was applied to each single cell to determine relative content of calcium (Ca), potassium (K), sodium (Na), chlorine (Cl), and phosphorus (P). Experimental chambers exhibited typical acute (0--9 min) and second phase (10--30 min) insulin secretion in association with suppression of glucagon release after 10 min. At 2 min an abrupt upward K spike in both alpha and beta cells was followed at 3--4 min with a 1.5- to 2-fold rise of Ca and a reciprocal decrease in K, Na, Cl, and P. From 3 to 30 min biphasic insulin secretion. Reduced alpha cell calcium after 6 min preceded suppression of glucagon secretion. After 2 min K related inversely to Ca content in both alpha and beta cells. These results could not be reproduced when D-galactose was substituted for D-glucose. We conclude that sequential changes of Ca content that are reciprocally related to K are predictive of beta cell insulin release and suppression of alpha cell glucagon secretion.     

p-Hydroxymercuribenzoic acid inhibits arachidonic acid esterification into two distinct pools and stimulates insulin release in intact rat islets

Activation of an islet phospholipase A2 may contribute to glucose-induced insulin release. In order to simulate the accumulation of the resultant hydrolytic products (arachidonic acid, AA; its lipoxygenase-derived oxygenation product 12-hydroxyeicosatetraenoic acid; and lysophospholipids) without many of the other concomitants of beta cell activation, we studied the effects on intact rat islets of p-hydroxymercuribenzoic acid (PHMB), which inhibits the reacylation of lysophospholipids with AA in other cell types. PHMB inhibited in a dose-responsive fashion (-90% at 500 microM) the incorporation of [3H]AA into a "basal" pool or pools whose release and reuptake mechanisms appeared to be largely energy- and Ca++-independent (resistant to inhibition by mannoheptulose, antimycin A or CoCl2); reciprocally, islets prelabeled with [3H]AA accumulated an increased amount of [3H]-12-hydroxyeicosatetraenoic acid (twice basal at 200 microM PHMB and three times basal at 500 microM) when reacylation of any [3H]AA released basally at 1.7 mM glucose was inhibited. PHMB also blocked (by up to 99% at 500 microM) the incorporation of [3H]AA into a functionally defined, glucose-stimulated compartment of fatty acid (tightly coupled to the islet 12-lipoxygenase) whose release and reuptake required metabolic energy and Ca++. It was also demonstrated that PHMB inhibited the esterification of [3H]AA (at low or high glucose concentrations) into specific phospholipids in islet membranes. In parallel with these alterations in lipid metabolism, PHMB caused rapid, potent and reversible increments in insulin release with a threshold concentration (about 25 microM) identical to that inhibiting AA fluxes. PHMB both initiated release (at 1.7 mM glucose) and potentiated the effects of islet fuels (16.7 mM glucose or 15 mM alpha-ketoisocaproic acid). Thus, pharmacologic manipulation of the AA reuptake mechanism is a new approach to unmask potential roles in insulin release of phospholipid hydrolysis products from different lipid pools and in the absence or presence of phospholipase A2 activation.     

Virus-induced alterations in cyclic adenosine monophosphate generation in hamster islets of Langerhans.

Inoculation of golden Syrian hamsters with Venezuelan encephalitis (VE) virus results in a sustained diminution in glucose-stimulated insulin release that is correctable by cyclic (c) AMP analogs and phosphodiesterase inhibitors. This suggested the importance of directly measuring cAMP content in VE-infected and control islets in response to insulin secretagogues. The basal cAMP content of VE-infected islets (0.14 +/- 0.02 pmol/micrograms islet DNA) was approximately half that of control islets (0.27 +/- 0.02 pmol/micrograms islet DNA) (P less than 0.05). In the presence of 10 microM glucagon (and 3 mM glucose), the rate of cAMP generation in VE-infected islets was only half that of control islets. With 10 mM alpha-ketoisocaproic acid, the rates of cAMP generation were indistinguishable between control and experimental groups. In response to 20 mM glucose and 3-isobutyl-1-methylxanthine (IBMX) (a phosphodiesterase inhibitor), cAMP generation in VE-infected islets was 81% (NS) of the control rate. When a more specific phosphodiesterase inhibitor, RO 20-1724, was used with 20 mM glucose, cAMP generation in the infected islets was only 44% (P less than 0.001) of the control value. Insulin secretion over the perifusion period paralleled the cAMP levels. In the presence of 10 mM alpha-ketoisocaproic acid, there was no difference in insulin secretion between VE-infected and control islets, while there was a statistically significant (P less than 0.05) difference with 10 microM glucagon or 20 mM glucose (in 1 mM RO 20-1724). These data point to a defect in the cAMP generation system of VE-infected islets, although additional factors involved in insulin secretion may also be impaired by the virus.     

Glucose and ATP levels in pancreatic islet tissue of normal and diabetic rats.

It has been suggested that the hyperglucagonemia observed in diabetic animals and man may be due to an impairment of glucose uptake and metabolism by the alpha-cells resulting in a decreased production of ATP. To test this hypothesis glucose, ATP, glucagon, and insulin were measured in pancreatic islets of normal and alloxan or streptozotocin diabetic rats. Two experimental approaches were used. In the first, the pancreas was perfused in vitro for assessing insulin and glucagon release due to 10 mM amino acids with and without 5 mM glucose. These perfusions were performed in the presence and absence of insulin. After perfusion, the pancreas was frozen and processed for analysis of islet glucose, ATP, insulin, and glucagon content. The second approach was to investigate the islet sucrose, urea, and glucose spaces together with ATP, insulin, and glucagon content in vivo in normal and in insulin-treated and untreated streptozotocin diabetic rats. Perfusion of the pancreas in vitro with 5 mM glucose resulted in higher glucose content of normal islets than in alloxan and streptozotocin diabetic islets. Similarly in the in vivo studies, the intracellular glucose space of the streptozotocin diabetic islets was 30% the value found in normals. In the in vivo experiments, despite the relatively small intracellular glucose space of alpha-cell islets, the ATP content of these islets was only 15-20% lower than the ATP content of normal islets. In the in vitro experiments, perfusion with glucose resulted in ATP contents of alpha-cell islets and of normal mixed alpha-beta-cell islets which were indistinguishable. However, the ATP content of alpha-cell islets was maintained for prolonged periods in the absence of glucose in contrast to mixed islets, composed primarily of beta-cells, in which the ATP level decreased by 45% when glucose-free medium was perfused for sustained periods. Finally, insulin infused in high concentrations or administered to the diabetic animal had no effect on the glucose spaces or the ATP contents of normal or alpha-cell islets. It can be calculated that in vivo the intracellular glucose level of islets from streptozotocin treated rats is approximately 15 mM. Since in normals an extracellular glucose concentration of this magnitude inhibits stimulated glucagon release completely, it would seem unlikely that a lack of intracellular glucose is the cause of the apparent glucose "blindness" of the alpha-cells in diabetes. In fact, in perfusion studies as little as 2.5 mM free intracellular glucose was sufficient to suppress glucagon secretion from diabetic alpha-cells. The results of the ATP measurements clearly eliminate a possible energy deficit of diabetic alpha-cells as cause of the apparent glucose resistance of alpha-cells.     

Inhibition of Na/Ca exchange stimulates insulin release from isolated rat pancreatic islets

Summary— Na/Ca exchange was recently shown to regulate cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic B-cell. The aim of the present study was to provide direct evidence that inhibition of the activity of the exchange may also increase insulin release. In the presence of extracellular Na+, caffeine stimulated ⁴⁵Ca outflow but did not increase insulin release from islets perifused in the presence of 2.8 mM glucose. By contrast, in the absence of extracellular Na+, caffeine almost failed to increase ⁴⁵Ca outflow and reversibly stimulated insulin release despite the fact that the absence of extracellular Na+ per se reduced basal insulin release. Similar findings were observed in islets perifused at a higher glucose concentration (8.3 mM) except that, in the presence of extracellular Na+, caffeine more markedly increased ⁴⁵Ca outflow and stimulated insulin release. Our data provide direct evidence that inhibition of Na/Ca exchange with resulting blockade of Ca²⁺ outflow may increase insulin release from the pancreatic B-cell under suitable experimental conditions.     

Rapid Transient Efflux of Phosphate Ions from Pancreatic Islets as an Early Action of Insulin Secretagogues

Anionic fluxes during the membrane realignments of stimulated insulin release have not been characterized previously although cations have been implicated in stimulus-secretion coupling. We have shown that a limited packet pulse of phosphate release ("phosphate flush") begins at the same time that the first phase of insulin secretion may occur. To demonstrate this phenomenon, we have prelabeled islets, obtained from rat pancreas by collagenase digestions, by incubation with [(32)P]orthophosphate. When such prelabeled islets are perifused with Krebs-Ringer bicarbonate containing 0.5 mg/ml D-glucose, a basal rate of efflux of radioactivity is established; transfer to perifusates containing 3.0 mg/ml D-glucose elicits an increased (32)P efflux within 1-2 min to peak values which are 7- to 21-fold greater than basal. The total duration of this "phosphate flush" approximates 10 min and exceeds the duration of the first phase of stimulated insulin secretion. With lesser concentrations of glucose, the flush exhibits dose-response relationships, and with 3 mg/ml glucose, a second flush can be elicited by restoring basal conditions and stimulating anew with 3 mg/ml glucose. The phenomenon is highly specific and can be reduplicated by other secretagogues (L-leucine) or sugars (D-mannose) which are also known to elicit insulin release but not by sugars which fail to affect insulin secretion (D-galactose, D-fructose, i-inositol, L-glucose). The efflux of radioactivity consists entirely of [(32)P]orthophosphate. Phosphate flush persists in phosphate-free media, Ca(++)-free media, and when insulin release is obtunded by adding Ni(++) (2 mM) to the perifusates. Thus, efflux of [(32)P]orthophosphate can be dissociated from insulin extrusion, and from net influx of ionic phosphate or calcium. Membrane stabilization with D(2)O or 1.0 mM tetracaine reversibly inhibits phosphate flush. Although the mechanism by which this effect occurs has not yet been established, the phosphate flush appears to constitute one of the earliest and hitherto unknown indices of the excitatory state in pancreatic islets.     

Survival of isolated human islets of Langerhans maintained in tissue culture.

Transplantation of human pancreatic islets to diabetic patients may require that donor islets be kept viable in vitro for extended time periods before transfer to the recipient. We have maintained isolated pancreatic islets obtained from the human cadaveric pancreas in tissue culture for 1-3 wk, after which we studied the structure and function of the islets. Electron micrographs of the cultured islets showed a satisfactory preservation of both beta-cells and alpha 2-cells. After culture for 1 wk, the islet oxygen uptake proceeded at a constant rate at a low glucose concentration (3.3 mM) and was significantly enhanced by raising the glucose concentration to 16.7 mM. Likewise, after culture for 1 wk, the islets responded with an increased insulin release when exposed to 16.7 mM glucose with or without added theophylline (10 mM). Islets cultured for 1-3 wk were able to incorporate [3H]leucine into proinsulin, as judged by gel filtration of acid-alcohol extracts. Glucagon release from the cultured islets was reduced significantly by 16.7 mM glucose alone, but stimulated by glucose (16.7 mM) plus theophylline (10 MM). It is concluded that viable pancreatic islets can be isolated from the pancreas of adult human donors and maintained in tissue culture for at least 1 wk without loss of the specific functions of the alpha 2- and beta-cells. It remains to be established whether such islets will survive and remain functionally competent after transplantation to human recipients.     

Drastic effects of cryoglobulin on blood cell counts,erythrocyte morphology and M-protein analysis

Abstract

Objective. Gamma-amino butyric acid (GABA) is an inhibitory neurotransmitter found widely in the central nervous system and some peripheral tissues such as the islets of pancreas. The aim of this study was to determine the time dependency of the effect of GABA on glucose-stimulated insulin secretion in isolated islets of rats. Materials and Methods. The collagenase digestion technique was used to isolate the islets from pancreata of male Wistar rats (200–250 g). Insulin secretion was assessed in islets exposed to different concentrations of glucose (8.3 and 16.7 mM), in the presence or absence of GABA (50 µM), for different incubation times and also pre-incubation of islets with GABA, 30 and 45 min before 8.3 mM glucose stimulation. Furthermore insulin secretion with different concentrations of glucose (8.3 and 16.7 mM), and in the presence or absence of different concentrations of baclofen, a GABA_B agonist and of saclofen, a GABA_B antagonist during 1 h of incubation was assessed. Insulin release was reported as mean ± SE μU/islet/min and p values of <0.05 were considered significant. Results. GABA with concentration of 50 µM inhibited the glucose stimulated insulin secretion during a 60 min incubation period. Baclofen had no significant effect on glucose-induced insulin secretion, while saclofen (100 µM) significantly increased 16.7 mM glucose-induced insulin secretion in 60 min of incubation (p < 0.05). Pre-incubation of islets with GABA for 45 min, before glucose stimulation, increased glucose-induced insulin secretion. Conclusion. GABA could have both stimulatory and inhibitory effects on glucose-stimulated insulin secretion, depending on the time of exposure and glucose concentration.     

Ionic and secretory response of pancreatic islet cells to minoxidil sulfate

Minoxidil sulfate is an antihypertensive agent belonging to the new class of vasodilators, the "K+ channel openers." The present study was undertaken to characterize the effects of minoxidil sulfate on ionic and secretory events in rat pancreatic islets. The drug unexpectedly provoked a concentration-dependent decrease in 86Rb outflow. This inhibitory effect was reduced in a concentration-dependent manner by glucose and tolbutamide. Minoxidil sulfate did not affect 45Ca outflow from islets perfused in the presence of extracellular Ca++ and absence or presence of glucose. However, in islets exposed to a medium deprived of extracellular Ca++, the drug provoked a rise in 45Ca outflow. Whether in the absence or presence of extracellular Ca++, minoxidil sulfate increased the cytosolic free Ca++ concentration of islet cells. Lastly, minoxidil sulfate increased the release of insulin from glucose-stimulated pancreatic islets. These results suggest that minoxidil sulfate reduces the activity of the ATP-sensitive K+ channels and promotes an intracellular translocation of Ca++. The latter change might account for the effect of the drug on the insulin-releasing process. However, the secretory response to minoxidil sulfate could also be mediated, at least in part, by a modest Ca++ entry.     

Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse B cells.

Glucose stimulation of insulin release involves closure of ATP-sensitive K+ channels, depolarization, and Ca2+ influx in B cells. Mouse islets were used to investigate whether glucose can still regulate insulin release when it cannot control ATP-sensitive K+ channels. Opening of these channels by diazoxide (100-250 mumol/liter) blocked the effects of glucose on B cell membrane potential (intracellular microelectrodes), free cytosolic Ca2+ (fura-2 method), and insulin release, but it did not prevent those of high K (30 mmol/liter). K-induced insulin release in the presence of diazoxide was, however, dose dependently increased by glucose, which was already effective at concentrations (2-6 mmol/liter) that are subthreshold under normal conditions (low K and no diazoxide). This effect was not accompanied by detectable changes in B cell membrane potential. Measurements of 45Ca fluxes and cytosolic Ca2+ indicated that glucose slightly increased Ca2+ influx during the first minutes of depolarization by K, but not in the steady state when its effect on insulin release was the largest. In conclusion, there exists a mechanism by which glucose can control insulin release independently from changes in K(+)-ATP channel activity, in membrane potential, and in cytosolic Ca2+. This mechanism may serve to amplify the secretory response to the triggering signal (closure of K(+)-ATP channels--depolarization--Ca2+ influx) induced by glucose.